Project Summary Although Landsteiner's discovery of ABO(H) blood group antigens over a century ago allowed predictions of red blood cell (RBC) compatibility prior to transfusion, in the 1930s several studies demonstrated that RBC transfusion itself can also induce additional RBC alloantibodies capable of causing incompatible transfusion reactions. RBC-induced alloantibodies against a variety of distinct RBC alloantigens can become particularly problematic in individuals who require repeat transfusions, often making it difficult, if not impossible, to find compatible RBCs. As a result, RBC alloantibody formation can significantly increase the mortality and morbidity of patients who require chronic transfusions. However, no current strategies exist to actively inhibit RBC-induced alloantibody formation following RBC transfusion therapy. This in part reflects a lack of understanding regarding key initiating factors that regulate RBC-induced alloantibody formation. In order to develop strategies with the potential to inhibit RBC-induced alloantibody formation, we used recently developed mouse models of RBC alloimmunization to define key initiating events required for the production of RBC alloantibodies following exposure to two disparate RBC alloantigens. Our preliminary results demonstrate that removal of marginal zone (MZ) B cells or macrophages within the MZ prevents alloantibody formation following exposure to two distinct RBC alloantigens. However, these unique RBC alloantigens appear to differentially induce MZ constituents to engage type 1 interferon (INF??) or toll-like receptor pathways. Furthermore, our preliminary data demonstrate that RBC alloantigens also possess the capacity to induce CD4+ T cell-independent or CD4+ T cell-dependent antibody formation following RBC transfusion. As previous studies suggest that marginal zone macrophages (MZM) and MZ B cells can work in a coordinated fashion to induce CD4+ T cell-independent or CD4+ T cell-dependent immune responses, these results suggest a critical link between MZM and MZ B cells in the generation of anti-RBC alloantibodies through both CD4+ T cell-independent and CD4+ T cell-dependent processes, depending on the type of RBC alloantigen presented. As a result, we hypothesize that MZ constituents play a central role in the initiation and orchestration of immune responses induced by RBC transfusion. To test this hypothesis, we propose the following specific aims: 1) Define the role of MZ B cells and MZM in IFN??-induced T cell-independent RBC alloantibody formation. 2) Define the role of MZM and MZ B cells in CD4+ T cell-dependent RBC alloantibody formation. We believe each of these aims provides a unique opportunity to identify common initiating factors key to the immune response to distinct RBC alloantigens. In doing so, these studies will provide valuable insight into potential targets for therapeutic mitigation of alloimmune responses to multiple RBC antigens in patients who require chronic transfusion therapy.